Platelet-Targeted Microfluidic Isolation of Cells

ABSTRACT

Methods and systems for isolating platelet-associated nucleated target cells, e.g., such as circulating epithelial cells, circulating tumor cells (CTCs), circulating endothelial cells (CECs), circulating stem cells (CSCs), neutrophils, and macrophages, from sample fluids, e.g., biological fluids, such as blood, bone marrow, plural effusions, and ascites fluid, are described. The methods include obtaining a cell capture chamber including a plurality of binding moieties bound to one or more walls of the chamber, wherein the binding moieties specifically bind to platelets; flowing the sample fluid through the cell capture chamber under conditions that allow the binding moieties to bind to any platelet-associated nucleated target cells in the sample to form complexes; and separating and collecting platelet-associated nucleated target cells from the complexes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/502,136, filed on Feb. 6, 2017, which is a 371 U.S. National of PCTApplication No. PCT/US2015/044375, filed on Aug. 7, 2015, which claimspriority from U.S. Provisional Application Ser. No. 62/034,522, filed onAug. 7, 2014, the contents of all of which are incorporated herein byreference in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under 5-U01-EB012493-04and EB002503-06A1 awarded by National Institutes of Health, and under125929-PF-14-137-01-CCE awarded by American Cancer Society. TheGovernment has certain rights in the invention.

TECHNICAL FIELD

This invention relates to the isolation of nucleated cells from fluidssuch as blood.

BACKGROUND

Metastasis, the spread and growth of tumor cells from the primary siteto distant organs, represents the most devastating and deadly attributeof cancer and is responsible for 90% of cancer deaths. Although asystematic understanding of metastasis biology is yet to be established,there is a growing recognition of the importance of circulating tumorcells (CTCs) as metastasis-initiating cells, which will provide apotential accessible source for early diagnosis, characterization andmonitoring of cancer progression. The reliable detection andnon-invasive isolation of CTCs and other nucleated cells from the bloodof cancer patients, however, remains technically challenging, not onlybecause of their extremely rare presence (as low as one in a billion ormore blood cells), but also due to the level of heterogeneity inbiophysical and biochemical properties.

In terms of size, CTCs and other rare nucleated cells can be as small as5 to 8 microns, e.g., the size of red blood cells, or 8 to 18 microns,which is about the same size as human white blood cells, or over 100microns in the form of CTC clusters. In terms of surface chemistry, theexpression of epithelial cell adhesion molecule (EpCAM), a biomarkerthat has been widely used to target CTCs and epithelial cells in generalin positive-selection methods has exhibited a large variation betweenclinical samples and can also be significantly down-regulated withcancer progression as a result of the epithelial-mesenchymal transition(EMT). Furthermore, tumor cells have been reported to actively interactwith host cells in the microenvironment, e.g., blood cells such as whiteblood cells and platelets, during the development of metastasis, whichmay make the detection and isolation of CTCs even more challenging.

SUMMARY

The present disclosure describes methods and systems of isolatingplatelet-associated nucleated target cells, such as circulatingepithelial cells, circulating tumor cells (CTCs), circulatingendothelial cells (CECs), circulating stem cells (CSCs), neutrophils,and macrophages, from sample fluids, e.g., biological fluids, such asblood, bone marrow, plural effusions, and ascites fluid, using bindingmoieties that specifically bind to platelets. The methods includeflowing the sample fluid including the platelet-associated target cells,e.g., CTCs, through a chamber, e.g., a cell capture chamber, underconditions that allow the binding moieties to bind to any target cells,e.g., CTCs, e.g., platelet-coated CTCs, in the sample to form complexes;and then separating and collecting the target cells from thesecomplexes, thereby isolating the target cells from the sample fluid.

In certain embodiments, the methods are performed using systems, forexample, one- or two-stage microfluidic systems, designed to achieveplatelet-targeted target cell capture. In some embodiments, the fluid,e.g., blood, sample can be first processed through a microfluidicdebulking device to remove free, unbound platelets and red blood cells(RBCs) before the sample fluid passes through the cell capture chamber.The debulking device can include one or more arrays of microposts toimplement hydrodynamic size-based sorting. The resulting sample fluidcontaining target cells and white blood cells (WBCs) can then beprocessed through the cell capture chamber functionalized withanti-platelet antibodies for high-throughput capture ofplatelet-associated target cells such as CTCs. In some embodiments, thecell capture chamber can include a mixing structure that enhances theinteraction of any platelet-associated target cells in the sample fluidwith the platelet antibodies. In some embodiments, such a mixingstructure is embodied as a so-called “herringbone” micromixer.

In general, the disclosure features methods for isolatingplatelet-associated nucleated target cells, e.g., circulating epithelialcells, CTCs, CECs, CSCs, neutrophils, and macrophages, from a samplefluid as described herein. The methods include obtaining a cell capturechamber including a plurality of binding moieties bound to one or morewalls of the chamber, wherein the binding moieties specifically bind toplatelets; flowing the sample fluid through the cell capture chamberunder conditions that allow the binding moieties to bind to anyplatelet-associated nucleated target cells in the sample to formcomplexes; and separating and collecting target cells from the complexesthereby isolating the target cells from the sample fluid.

In some implementations, the methods described herein can furtherinclude treating the sample fluid with a platelet inhibitor prior toflowing the sample fluid through the cell capture chamber, wherein theplatelet inhibitor inhibits unbound platelets from adhering to othercells. For example, other cells can be platelets, red blood cells,and/or white blood cells. In various implementations, the plateletinhibitor can be theophylline, adenosine, dipyridamole, Argatroban,and/or prostaglandin I2.

In any of the embodiments described herein, the binding moieties can beantibodies that bind specifically to platelets.

In some implementations, the methods can further include selectivelydepleting unbound platelets from the sample fluid while maintainingplatelet-associated nucleated target cells in the sample fluid beforeflowing the sample fluid through the cell capture chamber. For example,the platelet depletion can be performed in a microfluidic device thatincludes a channel containing an array of microposts to implementdeterministic lateral displacement. For example, the microposts can bearranged in a plurality of rows, wherein the microposts are spaced apartwithin a row by a distance of about 30 to about 60 microns, e.g., about35 microns to about 56 microns, subsequent rows are spaced apart from aprevious row by a distance of about 5 microns to about 15 microns, e.g.,about 5.6 microns to about 9.0 microns, and wherein the microposts ineach subsequent row are offset laterally from microposts in a previousrow by a distance less than the spacing between the microposts withinthe row.

In other implementations, the platelet depletion is performed in amicrofluidic device using centrifugal or inertial forces, or both or bydensity gradient centrifugation.

In some embodiments, the cell capture chamber and the microfluidicdevice can be both located on a single substrate or they can be locatedon separate substrates and are in fluid connection via a conduit. Insome implementations, the cell capture chamber includes a plurality ofchevron structures on an internal surface thereof arranged to createmicrovortices within the sample fluid.

In some embodiments designed for selective removal of the target cells,the binding moieties are bound to nanostructures that include a firstmember of a binding pair, wherein one or more internal surfaces of thecell capture chamber are bound to a layer of gelatin functionalized witha plurality of second members of the binding pair, and wherein thenanostructures are bound to a top layer of the gelatin by a bindinginteraction of the first and second members of the binding pair. In someimplementations of these methods, the platelet-associated nucleatedtarget cells are bound to the nanostructures by the binding moieties andthe platelet-associated nucleated target cells are isolated by releasingthe nanostructures from the gelatin by melting the gelatin at anincreased temperature. Alternatively, the target cells can be isolatedby releasing the nanostructures from the gelatin by applying a localizedshear stress to the gelatin layer or by using a light-targetedphotothermal effect.

In another aspect, the disclosure features a systems, e.g., two-stagemicrofluidic systems, for isolating platelet-associated nucleated targetcells such as CTCs from a sample fluid. These systems include a firstchamber, a second compartment, and a conduit fluidly connecting the twocompartments. In particular, the conduit fluidly connects the productoutlet of the first chamber to the inlet of the second chamber.

In these systems, the first compartment includes a microchannel havingan inlet, a waste outlet, a product outlet, and an array of micropostsarranged between the inlet and the outlets, wherein the microposts arearranged in rows and spaced apart by a distance that enables red bloodcells and unbound platelets to flow through the device to a waste outletand to cause platelet-associated nucleated target cells to be laterallydisplaced by the array of microposts to a product outlet, wherein themicroposts in each subsequent row are offset laterally from micropostsin a previous row by a distance less than the spacing between themicroposts within the row.

The second chamber includes a microchannel having an inlet and anoutlet, wherein fluid flows from the inlet to the outlet through themicrochannel, and a plurality of grooves defined in and arranged on aninternal surface of one or more walls, floor, and ceiling of themicrochannel to create microvortices within the sample fluid; andbinding moieties fixed to at least one internal surface, wherein thebinding moieties specifically bind to platelets. For example, thebinding moieties can be antibodies that bind specifically to platelets.

In various implementations of these systems, the microposts are spacedapart within a row by a distance of about 30 microns to about 60microns, e.g., about 35 microns to about 56 microns, and subsequent rowsare spaced apart from a previous row by a distance of about 5 microns toabout 15 microns, e.g., about 5.6 microns to about 9.0 microns. Indifferent embodiments the first chamber and the second chamber are bothlocated on a single substrate or they can be located on separatesubstrates and are in fluid connection via the conduit.

In some embodiments, the grooves in the second chamber include an apexand two arms connected to the apex to form a V-shape, and the groovesare arranged such that the sample fluid flows past the arms towards theapex.

In certain implementations of these systems, the binding moieties arebound to nanostructures that comprise a first member of a binding pair,wherein one or more internal surfaces of the second chamber are bound toa layer of gelatin functionalized with a plurality of second members ofthe binding pair, and wherein the nanostructures are bound to a toplayer of the gelatin by a binding interaction of the first and secondmembers of the binding pair.

As used herein, the term “binding moiety” means any molecule or agentthat can adhere to a target such as a particle, molecule, or cell.Binding moieties include, for example, members of a ligand binding pair,antibodies, aptamers, or nucleic acid molecules. Some binding moietiesbind specifically to a target molecule or agent, such as a receptor or acell surface marker on a cell surface, and some bind non-specifically toa variety of targets that may share a common feature.

As used herein, the term “specific binding” means that a binding moietybinds selectively and preferentially to a particular target, such as aparticle, molecule, or cell, e.g., to a molecule on the surface of acell, in a sample including other particles or molecules.

Compared with prior CTC sorting techniques that rely on specialbiophysical and/or biochemical properties of cancer cells, the methodsand systems described herein focus on active cell-cell interactionsduring cancer metastasis, which is not limited by the size, cancer type,or the expression level of tumor surface antigens. More importantly, thenew methods and systems enable the isolation of a very specialsubpopulation of nucleated target cells, such as CTCs, which arecorrelated with high metastatic potential, but are difficult to targetby other techniques, and thus provide valuable information for bothearly cancer diagnostics and a better understanding of how cancerspreads.

In particular, one benefit of the new methods and systems describedherein is that they do not rely on specific biomarkers on the surface ofthe target cells. Many current techniques rely on the expression ofepithelial cell adhesion molecule (EpCAM), a biomarker that has beenwidely used to target epithelial cells and CTCs in positive-selectionmethods. However, EpCAM expression varies widely between clinicalsamples and can also be significantly down-regulated with cancerprogression as a result of the epithelial-mesenchymal transition (EMT).The present methods and systems overcome this difficulty with priormethods.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. The details of one or more embodiments of the inventionare set forth in the accompanying drawings and the description below.Other features, objects, and advantages of the invention will beapparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic diagram of tumor metastasis viaplatelet-associated CTCs.

FIG. 1B is a schematic diagram of one embodiment of a two-stagemicrofluidic platform designed to achieve platelet-targeted CTC capture.

FIG. 2A is a schematic diagram of one embodiment of a debulking chip.

FIG. 2B is a schematic diagram of one embodiment of the micropost arrayon the debulking chip.

FIG. 3 is a schematic diagram of one embodiment of a microvortex“herringbone” chip.

FIGS. 4A to 4D are a series of schematic diagrams of a flow pattern ofparticles, e.g., cells or clusters of cells, flowing through themicrovortex “herringbone” chip of FIG. 3.

FIG. 5 is a graph showing CTC counts of blood samples from metastaticlung patients that were captured with EpCAM and CD41 antibodies,respectively.

FIG. 6 is a graph showing counts of CTCs in the form of single cells,clusters of 1 or 2 WBCs, and clusters of greater than 2 WBCs.

FIG. 7 is a graph showing reduced platelet-leukocyte aggregate formationwith the addition of EDTA and prostaglandin I2.

FIGS. 8A and 8B are heat maps of WBCs captured on a microvortex“herringbone” chip for non-treated (8A) and prostaglandin I2-treated(8B) blood samples.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The interaction between tumor cells and platelets is thought to play arole in blood-borne metastasis, as shown schematically in FIG. 1A. Themost compelling evidence is the inhibition of metastasis by plateletdepletion and the restoration of metastatic potential by plateletreconstitution in several independent mouse studies. The uniquecapability of malignant tumor cells to activate and aggregate plateletson a surface, a process known as tumor cell-induced platelet aggregation(TCIPA), confers a number of advantages to the tumor in successfulmetastasis. The platelets might not only contribute to the vascularremodeling process in angiogenesis, but may also significantly enhancetumor cell survival in the bloodstream by shielding them against theshear stress and immune surveillance.

According to the new methods and systems described herein, a broad setof adhesion receptors on platelets can be used to isolateplatelet-associated nucleated target cells, such as epithelial cells,CTCs, neutrophils, and macrophages from sample fluids such as blood,e.g., whole blood. In addition, recent research on the signaling betweenplatelets and tumor cells revealed that the platelet-derived growthfactors/cytokines help induce the epithelial-mesenchymal transition(EMT) and further enhance the metastatic potential. As a result, the newmethods and systems are able to isolate CTCs that have an enhancedmetastatic potential and can thus provide better diagnostic informationthan other CTCs that are not associated with platelets.

Methods of Isolating Target Cell-Platelet Clusters from Sample Fluids

The present methods and systems take advantage of the unique interplaybetween certain nucleated target cells, e.g., CTCs, and platelets andutilize platelets that are associated with such target cells, e.g.,bound to, e.g., coated on, the surface of the target cells, as aubiquitous, cell-based markers to target and isolate these target cellsfrom sample fluids, such as blood, e.g., whole blood.

In general, the new methods use a chamber including a plurality ofbinding moieties bound to one or more walls of the chamber, wherein thebinding moieties specifically bind to platelets; flowing the samplefluid including the target cells, if any, through the chamber underconditions that allow the binding moieties to bind to anyplatelet-associated target cells in the sample to form complexes; andseparating and collecting target cells from the complexes therebyisolating the target cells from the sample fluid. Further, the methodscan also include steps of selectively depleting unbound platelets and/orother contaminating cells, such as red blood cells (RBCs) from thesample fluid while maintaining platelet-associated target cells in thesample fluid before flowing the sample fluid through the chamber and/orusing mixing in the chamber to enhance contacts between the plateletsand the binding moieties.

Since TCIPA is a general phenomenon originating from the intrinsicinteraction between tumor cells and host microenvironment, theplatelet-targeted methodology has the potential to detect and isolate abroad spectrum of nucleated target cells in blood-borne metastasisindependent of tumor membrane epitopes.

To reduce WBC contamination and improve CTC purity, platelet inhibitorscan be added to the blood sample prior to testing to stabilize theblood. A variety of platelet inhibitors can be used, such astheophylline, adenosine, dipyridamole, Argatroban, and prostaglandin I2,for blood stabilization. A combination of EDTA with prostaglandin I2 isparticularly effective at inhibiting the formation of platelet-leukocyteaggregates (PLA), and can reduce the number of contaminating WBCs in ablood sample by 90%.

Binding Moieties for Binding to Platelet-Associated Target Cells

Various binding moieties can be used to bind to the platelets in thesample fluid. For example, a variety of antibodies are known to targetdifferent platelet surface receptors, including CD41 and CD61 (subunitsof integrin α2bβ3); CD42b and CD42c (the major receptors for vonWillibrand factors (vWF); glycoprotein VI (GP VI) (the collagenreceptor); and the thrombopoietin receptor (TPO-R). Of these anti-CD41antibodies can be used effectively, as they have a high captureefficiency for platelets and low non-specific binding.

The binding moieties can be functionalized onto a solid support, such asPDMS or glass surfaces using various methods, such as silane chemistry.Different silane precursors with amine, aldehyde, or thiol terminalgroups can be crosslinked with oxygen-plasma treated PDMS/glasssurfaces, which can further be conjugated to different binding moieties.In a particular example, a solid support in the form of a microfluidicchannel is modified with 3-mercaptopropyl tri-methoxy silane, followedby the addition of N-y-maleimidobutyryloxy succinimide ester as thelinker, and finally conjugated to NeutrAvidin. Any biotinylated bindingmoieties, such as biotinylated platelet antibodies, can then be easilyfunctionalized onto the device through avidin-biotin chemistry.

Flowing the Sample Fluid Through the Chamber with Binding Moieties

After surface functionalization, the devices are typically blocked withan agent to avoid non-specific binding, e.g., with a sufficient amountof bovine serum albumin (BSA), e.g., 3% BSA. Thereafter a specificvolume, e.g., 1 to 10 mL, e.g., 2, 3, 4, 5, or 6 mL, of a sample fluid,e.g., blood, e.g., whole blood, or buffy coat, are flowed into thesystem. The sample fluids are typically processed at flow rates of 1-5mL/hour, e.g., 1, 2, 3, or 4 mL/hour, at room temperature under a setpressure, e.g., 0.03 to 0.15 psi, e.g., 0.05, 0.075, or 0.1 psi.

Debulking the Sample Fluid to Remove Unbound Platelets

Because of the abundance of platelets in whole blood (˜10⁵/μL) and thepotential effect on saturating the binding moieties, the blood samplecan be first processed to remove free, unbound platelets. In addition,it is helpful to remove some or most of the red blood cells (RBCs). Thedebulking can be accomplished, for example, with density gradientcentrifugation or microfluidic-based cell separation.

Density gradient centrifugation is generally performed with bloodsamples collected in CPT tubes, which contain anticoagulant togetherwith a polyester gel and a density gradient liquid. Aftercentrifugation, the nucleated cells can then be harvested by carefullypipetting them from the liquid interface with minimal contamination fromred blood cells and platelets.

The microfluidic-based blood debulking can be accomplished withdifferent designs and techniques known in this field. For example,inertial forces can be utilized in curved, e.g., serpentine,microfluidic channels to differentially focus and sort cells based ontheir sizes by centrifugal or inertial forces (e.g., inertial focusing),or both (see, e.g., U.S. Pat. No. 8,807,879). In addition, hydrophoreticfiltration and/or acoustic standing waves can also be utilized to sortcells of different sizes.

In another implementation, the unbound platelets can be removed from thesample fluid using a microfluidic debulking device. Such devices can becomposed of one or more arrays of microposts to implement hydrodynamicsize-based sorting, as described in further detail below. In general,these microfluidic debulking device uses hydrodynamic size-based sortingto achieve low shear microfluidic debulking of whole blood. RBCs,platelets, and plasma proteins are discarded, whereas nucleated cells(WBCs and CTCs) are retained and presented to the second stage fortarget cell, e.g., CTC, capture. The operational principle ofmicrofluidic debulking is based on hydrodynamic size-dependentdeterministic lateral displacement, in which coincident flow ofcell-containing and cell-free solutions through an array of micropostsleads to rapid size-based separation (see, e.g., U.S. Pat. Nos.8,986,966 and 8,585,971). See also, Ozkumur et al., “Inertial Focusingfor Tumor Antigen-Dependent and -Independent Sorting of Rare CirculatingTumor Cells,” Science Translational Medicine, 5(179):179ra47 (DOI:10.1126/scitranslmed.3005616) (2013).

By optimizing the array configuration (including the gap or spacebetween the microposts and the shift between adjacent rows) and flowrate, one can achieve over a 5-log depletion of free, unbound plateletswhile maintaining most of the nucleated cells. The fluid productcontaining nucleated target cells and white blood cells (WBCs) can thenbe directed to the chamber that contains the platelet-specific bindingmoieties as described herein.

Mixing the Sample Fluid to Enhance Binding Interactions

To enhance the interaction of any platelet-associated target cells inthe sample fluid with the binding moieties in the chamber, one caninclude a mixing structure within the chamber. For example, differentchannel structure designs, including zigzag, serpentine, or twistedchannels, can be utilized for passive mixing. In addition, it is alsopossible to further enhance the mixing performance by incorporatingactive micro-mixing enabled by acoustic, pressure perturbation, ordielectrophoretic techniques.

One useful design is a so-called “herringbone” micromixer included onone or more of the internal walls, floor, or ceiling of the chamberfunctionalized with platelet antibodies for high-throughput capture ofplatelet-associated target cells such as CTCs (see, e.g., PCTApplication No. WO 2010/036912). In general, 1-10 mL of a sample fluid,e.g., buffy coat or debulked blood, are flowed into the system. Thesample fluid with continuous rocking was typically processed through thedevice at the flow rate of 1 to 2 mL/hr at room temperature under0.03-0.15 psi, e.g., 0.05, 0.075, or 0.1 psi.

Processing the Target Cells Captured in the Chamber

All cells captured in the cell capture device can be processed foridentification, e.g., with a staining assay, e.g., a four-color stainingassay, for simultaneous target cell identification and plateletcharacterization. The captured cells that are positive for tumor markers(e.g., EpCAM, cytokeratin, epithelial growth factor receptor (EGFR),human epidermal growth factor receptor 2 (HER-2), cadherin-11, and 4′,6-Diamidino-2-Phenylindole (DAPI)), and optionally those that are alsonegative for hematopoietic markers (such as CD45, CD14, CD16) are scoredas CTCs. Reliable CTC capture can be achieved using the new methods withcounts ranging from 0.4 to 8.5 CTCs/mL of whole blood samples. Othertarget cells can be identified using other markers. For example,epithelial cells can be detected with CD24, CD133, and CD326;neutrophils with CD15, CD16, and CD66b; and macrophages with CD11b,CD68, and CD163. In addition, circulating endothelial cells (CECs) canbe identified with CD34 and CD146 and circulating stem cells (CSCs) canbe identified with CD44, CD90, and ALDH1.

When CTCs are captured on microfluidic devices using prior techniques,e.g., devices functionalized with anti-epithelial marker antibodies,such as EpCAM antibodies, the results reveal consistently lower positivehits that then present methods. See FIG. 5, which as discussed infurther detail in Example 2 below, shows that the cell capture systemsdescribed herein are capable of reliable capture of CTCs in the form ofsingle cells or clusters from metastatic cancer patients with bothepithelial (lung, breast) and non-epithelial (melanoma) tumor origins.The higher CTC counts from the present platelet-targeted approach aredue, in part, to the new methods' capability to capture CTCs from thelung and other epithelial tumor types that might lose their epithelialnature and thus become more difficult to target by anti-epithelialmarker antibodies, such as EpCAM antibodies.

Prior EpCAM-targeted approaches generally yield CTCs with limitedassociated platelets, while the CTCs captured using the new methodsdescribed herein exhibit a wide range of platelet coverage, including aspecial subpopulation of CTCs that were completely coated withplatelets/fibrin. These platelet-coated CTCs have been hypothesized ashigh metastatic potential precursors because of platelet-enhancedtumor-cell survival and proliferation, but are very difficult to captureby conventional positive selection methods targeting tumor surfaceantigens. Quantitative imaging methods can be used to furtherinvestigate the correlation between CTC phenotype and plateletdistribution. Combined with controlled cell release strategies that wererecently developed by our group, it is also possible to achievesingle-cell level genotypic studies that are expected to further advanceour knowledge about cancer metastasis.

Once the platelet-associated CTCs are captured using the new methods,breast cancer CTCs can be identified using the same protocol as lungcancer samples, while melanoma CTCs are identified by staining with anantibody or antibody cocktail that targets melanoma specific antigens(e.g., any one or more of anti-CSPG4, MCAM, TYRP1, and α-SMAantibodies).

Purification of Captured Target Cells Through Selective Release

In some sample fluids such as whole aged blood there may be a largepopulation of platelet-associated WBC cells that are isolated by the newmethods along with the platelet-associated target cells.

In some embodiments, the new methods can include steps to selectivelyrelease target cells such as CTCs bound to the chamber that contains thebinding moieties. For example, as described in International ApplicationPublication No. WO 2014/121204, the cell capture chamber can includebinding moieties that are bound to nanostructures that themselvesinclude a first member of a binding pair, wherein one or more internalsurfaces of the chamber are bound to a layer of gelatin functionalizedwith a plurality of second members of the binding pair, and wherein thenanostructures are bound to a top layer of the gelatin by a bindinginteraction of the first and second members of the binding pair. Thetarget cells (bound via their associated platelets to the bindingmoieties bound to the nanostructures) can then be released from thechamber, via either of two release mechanisms.

In the first release mechanism, the target cells such as CTCs can beisolated and removed from the cell capture chamber by releasing thenanostructures from the gelatin by melting the gelatin at an increasedtemperature. By increasing temperature, e.g., over 30° C., e.g., 37° C.,captured target cells can be released in a bulk fashion. Alternatively,in the second release mechanism, the target cells can be released fromthe gelatin by applying a localized shear stress the gelatin layer. Byincreasing a localized shear stress in the gelatin, e.g., by applying afrequency-controlled force with a vibrating device, e.g., a microtipdevice described, for example, in PCT WO 2014/121204 single cells can beselectively released from the cell capture chamber.

In addition to single CTCs (about 55%), the new methods also isolatedifferent sized cell clusters on the platelet-targeted platforms. Indeedover 40% of captured CTCs were in the form of CTC/WBC clusters. See FIG.6, which shows that about 30% of CTCs were clustered with 1 or 2 WBCs,and about 15% of CTCs were clustered with more than 2 WBCs. The numberof interacting WBCs increased with the platelet coverage around CTCs.The capability of the current approach to target both single CTCs andCTC/WBC clusters, the latter of which are very difficult to isolatethrough conventional affinity based positive or negative selectionmethods, would enable a comprehensive characterization of cancermetastasis through non-invasive blood biopsy, and also open up newopportunities in downstream applications, such as CTC culture, as aresult of the improved CTC survival/proliferation in the specializedmicroenvironment.

The ability to isolate CTC/WBCs provides for isolation of a cellpopulation that would otherwise be lost in other prior CTC isolationtechniques. For example, negative-selection CTC isolation techniquestarget WBCs with magnetic material using antibody-based binding. Inthese methods, the magnetic particles are functionalized to bindspecifically to WBCs and are introduced to the fluid sample prior toentering the microfluidic system. The fluid sample then enters a“deflection channel” subject to a magnetic gradient oriented to deflectthe magnetic particles, and any cells bound to them, in a certaindirection. When the fluid sample containing target cells/particles boundto one or more magnetic particles is introduced into a deflectionchannel, the magnetic force created by the nearby magnets pulls themagnetic particles (and the attached cells/particles) in a direction ofthe magnetic gradient. In the case of negative-selection CTC isolation,this separation diverts the WBCs, along with any other particles/cellsattached to the WBCs, to the “waste” channel and isolates non-bound CTCsin the “product” channel. This method results in the loss of any CTCsbound to WBCs, which in turn, are bound to the magnetic particles, tothe waste channel.

The two-stage microfluidic platforms described herein do not suffer fromthe same shortcomings.

Microfluidic Systems to Isolate Target Cells from Sample Fluids

As noted above, the methods for isolating platelet-associated nucleatedtarget cells, such as CTCs, from sample fluids employ a cell capturechamber that contains a plurality of binding moieties bound to one ormore walls, floor, and/or ceiling of the chamber, wherein the bindingmoieties specifically bind to platelets. The fluid sample including thetarget cells is flowed through the chamber under conditions that allowthe binding moieties to bind to any platelet-associated target cells inthe sample to form complexes. This process provides for the separationand collection of target cells, such as CTCs, from the complexes therebyisolating target cells from the sample fluid. Furthermore, as shown inFIG. 1B, the systems can include an upstream component (first stage 110)that selectively depletes unbound platelets and other cells, e.g., redblood cells (RBCs) from the sample fluid while maintainingplatelet-associated CTCs in the sample fluid before flowing the samplefluid through the cell capture chamber (second stage 120).

As shown in FIG. 1B, the input fluid to the system can be whole blood130. The first stage 110 separates unbound platelets and other cells,e.g., RBCs, 140 from the nucleated cells (150) that continue through thesystem to enter the platelet-targeted capture chamber (120).

Stage One—Debulking Device to Remove Red Blood Cells and Platelets fromSample Fluid

For the first stage, the overall system can include a debulking device200, e.g., a microfluidic device composed of one or more arrays ofmicroposts to remove RBCs and free platelets from whole blood throughhydrodynamic size-based sorting that allows the smaller platelets andRBCs to exit the device as waste while passing the larger WBC and CTCand cell clusters to the second stage, which is the cell capturechamber.

As shown in FIG. 2A, the first stage system can be manufactured on asingle chip 200. In some embodiments, the chip can include an inlet 202,one or more arrays of microposts 205 (two in the present figure), and anoutlet 203.

As shown in FIG. 2B, the first stage in the system can be a hydrodynamicsorting device for the separation of cells using an array of microposts205 that selectively allow passage of particles based on their size,shape, or deformability. The size, shape, or deformability of the spacesin the array of microposts determines the types of cells that can passthrough the array. Two or more arrays of microposts can be arranged inseries or parallel, e.g., to remove cells of increasing sizesuccessively. For a description of such microfluidic systems, see, e.g.,U.S. Pat. Nos. 8,986,966, 8,585,971 and Ozkumur et al. (2013), supra.

A variety of micropost 210 sizes, geometries, and arrangements can beused in the devices described herein. Different shapes of microposts,e.g., those with circular, square, rectangular, oval, or triangularcross sections, can be used. The size of the gaps 220 between themicroposts and the shape of the microposts can be optimized to ensurefast and efficient separation. For example, the size range of the RBCsis on the order of 5-8 μm, and the size range of platelets is on theorder of 1-3 μm. The size of all WBCs is greater than 10 μm. Takingthese dimensions into consideration, while larger gaps between themicroposts increase the rate at which the RBCs and the platelets passthrough the array, increased gap size also increases the risk of losingWBCs. Smaller gap sizes ensure more efficient capture of WBCs, but alsoa slower rate of passage for the RBCs and platelets. Depending on thetype of application different geometries can be used. For the presentmethods, micropost diameters of about 10 to 30 μm, e.g., about 15 to 24μm, e.g., 10, 12, 15, 17, 20, 22, 24, 25, or 27 μm are useful. Gaps orspaces between the microposts of about 10 to 40 μm, e.g., 20 to 32 μm,e.g., 15, 20, 25, 30, 35, or 40 μm are effective.

Arrays of microposts can be manufactured by various methods. Forexample, an array of microposts can be formed by molding,electroforming, etching, or drilling a substrate such as a glass, ametal, or a polymer. For example, simple microfabrication techniqueslike poly(dimethylsiloxane) (PDMS) soft lithography, polymer casting(e.g., using epoxies, acrylics, or urethanes), injection molding,polymer hot embossing, laser micromachining, thin film surfacemicromachining, deep etching of both glass and silicon, electroforming,and 3-D fabrication techniques such as stereo lithography can be usedfor the fabrication of the channels and array of microposts of devicesdescribed herein. Most of these processes use photomasks for replicationof micro-features.

For feature sizes of greater than 5 μm, transparency based emulsionmasks can be used. Feature sizes between 2 and 5 μm may requireglass-based chrome photomasks. For smaller features, a glass basedE-beam direct write mask can be used. The masks are then used to eitherdefine a pattern of photoresist for etching in the case of silicon orglass or define negative replicas, e.g., using SU-8 photoresist, whichcan then be used as a master for replica molding of polymeric materialslike PDMS, epoxies, and acrylics. The fabricated channels may then bebonded onto a rigid substrate like glass to complete the device. Othermethods for fabrication are known in the art and the device describedherein can be fabricated from a single material or a combination ofmaterials.

A specific first-stage hydrodynamic sorting chip was designed andfabricated deep reactive ion etching on silicon wafers. The chip wassealed with anodically bonded glass cover to form the microfluidicdebulking component. A custom polycarbonate manifold was used to formthe fluidic connections to the substrate and to the second-stage cellcapture chamber.

Stage Two—Cell Capture Chambers

In various embodiments, the cell capture chamber can be a simplemicrofluidic channel that is functionalized on one or more walls and/orthe floor with the platelet binding moieties, or can be more elaborateand include a mixing structure to enhance and increase the number ofcontacts of the platelet-associated CTCs in the sample fluid with theplatelet binding moieties.

As shown in FIG. 3, the cell capture chamber can be formed as amicrovortex-generating “herringbone” chamber (or “chip”) functionalizedwith anti-platelet antibodies for high throughput capture ofplatelet-associated CTCs (see e.g., PCT Application Publication No. WO2010/036912). In this embodiment, the chamber is formed as amicro-channel formed in a microfluidic device in which grooves (orprotrusions) are formed extending into (or out of) the walls of themicro-channel to create flow patterns in fluid flowing through thechannel that promote interactions between any cells suspended in thefluid and the inner surfaces of the walls of the channel. The increasedinteractions can lead to an increase in a number of CTCs captured in thechannel, and consequently, in the overall capture efficiency of themicrofluidic device. In this embodiment, the capture efficiency of themicrofluidic device is defined as a ratio of a number of CTCs capturedin the channel and a total number of cells flowed through the channel.

The efficiency can further be increased by tailoring structural featuresof the microfluidic device including, for example, device substratematerial, channel and groove dimensions, and the like, as well as fluidflow parameters such as flow rates based on types of particles and thetypes of fluids in which the particles are suspended.

An example of such a microfluidic device manufactured using softlithography techniques is described with respect to FIG. 3. As describedbelow, platelet-associated CTCs are captured in the micro-channel of thecell capture chamber by forming grooves in one or more of a wall, floor,and/or ceiling of the micro-channel, coating the platelet bindingmoieties on the inner surfaces of the walls, floor, and/or ceiling ofthe micro-channel, and flowing the sample fluid through themicro-channel.

FIG. 3 illustrates a microfluidic device 300 having grooves 335, 340extending into an upper wall (or ceiling) of a channel 315 of the device300. In some embodiments, the cell capture chambers include protrusionsextending outward from the wall (e.g., V-shaped protrusions) rather thangrooves extending into a wall of the channel 315. In some embodiments, asymmetric groove 335 includes two arms, each spanning a length between afirst end 350 and the apex 345, and a second end 355 and the apex 345.In the illustrated embodiments, the angle α between the two arms is 90°.In some embodiments, the angle α between the arms ranges between 10° and170°. In some implementations, a microfluidic device 300 can include anupper substrate 305 bonded to a lower substrate 310, each of which canbe fabricated using an appropriate material. For example, the uppersubstrate 305 can be fabricated using an elastomer such as, for example,polydimethylsiloxane (PDMS), and the lower substrate can be fabricatedusing glass, PDMS, or another elastomer.

Alternatively, or in addition, the substrates can be manufactured usingplastics such as, for example, polymethylmethacrylate (PMMA),polycarbonate, cyclic olefin copolymer (COC), and the like. In general,the materials selected to fabricate the upper and lower substrates canbe easy to manufacture, for example, easy to etch, and can offer opticalproperties that facilitate ease of testing, for example, can beoptically clear, and can be non-toxic so as to not negatively affect thecells attached to the substrate. In addition, the materials arepreferred to exhibit no or limited auto-fluorescence. Further, thematerials can be easy to functionalize so that analytes can be attachedto the substrate. Furthermore, the materials can be mechanically strongto provide strength to the microfluidic device 300. The upper substrate305 can be securely fastened to the lower substrate 310, with amicro-channel formed between them, as described below.

In some implementations, the micro-channel 315 can have a rectangularcross-section including two side walls 320 and 325, and an upper wall330 formed in the upper substrate 305. Terms of relative location suchas, for example, “upper” and “lower” are used for ease of descriptionand denote location in the figures rather than necessary relativepositions of the features. For example, the device can be oriented suchthat the grooves are on a bottom surface of the channel or such that acentral axis of the channel extends vertically. Alternatively, thecross-section of the micro-channel 315 can be one of several shapesincluding but not limited to triangle, trapezoid, half-moon, and thelike. The lower substrate 310 can form the lower wall of themicro-channel 315 once bonded to the upper substrate 305. In someimplementations, the micro-channel 315 includes multiple grooves 335formed in the upper wall 330 of the micro-channel 315. Alternatively,the grooves 335 can be formed in any of the walls, and/or can be formedin more than one wall of the micro-channel 315. The grooves 335 can spanan entire length of a wall, or only a portion of the wall.

FIGS. 4A-4D are schematics illustrating particle suspensions flowingthrough a micro-channel having flat walls and another micro-channelhaving grooves formed in a wall. FIG. 4A shows a microfluidic device 400that includes a micro-channel 405 having a rectangular cross-section.The walls of the micro-channel 405 do not include grooves such as thosedescribed with respect to the microfluidic device 300, i.e., surfaces ofthe walls are flat. A particle suspension including platelet-associatedtarget cells 425 suspended in a fluid is illustrated flowing through themicro-channel 405. In contrast, FIG. 4B shows a similar suspensionflowing through the microfluidic device 300.

As the fluid flows past a herringbone pattern formed by arranginggrooves 335 in a column in the micro-channel 315, the grooves 335 in thepath of the fluid disrupt fluid flow. In some embodiments, dependingupon flow velocity and the dimensions of the grooves, specifically, forexample, a size of the grooves and an angle between the two arms of agroove, the disruption in the fluid flow leads to a generation ofmicrovortices in the fluid. The microvortices are generated because thegrooves induce fluid flow in a direction that is transverse to aprincipal direction of fluid flow, i.e., the axial direction. In someembodiments, although microvortices are not generated, the grooves 335,340 induce sufficient disruption to alter the flow path of portions ofthe fluid to increase wall-particle interactions.

Without any grooves, as shown in FIG. 4C, the platelet-associated targetcells 425 suspended in the fluid travel through the flat micro-channel405 in a substantially linear fashion such that only those particles 425near the edges of the flow field (e.g., immediately adjacent to thewalls of the micro-channel 405) are likely to interact with antibodiesbound to the micro-channel 205 walls. In contrast, as shown in FIG. 4D,flowpaths of the platelet-associated target cells 425 traveling past theherringbone groove patterns experience disruption by the microvorticesin the fluid, increasing the number of interactions between the cellsand the antibodies bound to the walls and/or grooves. The microvorticesare affected by the structural features of each groove 335 formed in theupper wall 315 of the microfluidic device 300.

The second-stage herringbone chips can be made of PDMS bonded to glasssubstrates using soft lithography techniques as previously described.The chip surface can then be functionalized with anti-CD41 antibody(Abcam) using avidin-biotin chemistry.

The first-stage hydrodynamic sorting chip and the second-stageherringbone chip can be manufactured on a single chip, or can bemanufactured as separate chips and connected by a conduit, such asplastic or other tubing.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1—CTC Isolation from Clinical Samples

The present methods were tested to determine their efficacy in isolatingCTCs from different types of cancers.

Patients with advanced lung, breast, and melanoma cancer were recruitedaccording to a protocol approved by the institutional review board(IRB). All specimens were collected into Vacutainer® (Becton-Dickinson)tubes containing the anticoagulant EDTA and were processed through themicrofluidic chips within 4 hours of blood draw. Additional plateletinhibitors, such as theophylline, adenosine, dipyridamole, Argatrobanand prostaglandin I2, were added immediately after blood collection.Fixed blood samples were collected directly in Cyto-Chex® BCT tubes(Streck).

Samples were run on a two-stage microfluidic system described above. Inparticular, the first-stage hydrodynamic sorting chips were fabricatedwith deep reactive ion etching on silicon wafers. The chip was sealedwith anodically bonded glass cover to form the microfluidic chamber. Acustom polycarbonate manifold was used to form the fluidic connectionsto the microchip. We tested two different array configurations with gapsbetween microposts of 20 or 32 μm. An array with 20-μm gaps retainsvirtually all nucleated cells with minimal contaminating RBCs, but has acutoff for cells larger than 21 μm and may therefore lose large CTCs orCTC clusters. In contrast, an array with 32-μm gaps has an extendedoperating range for cells between 8 and 30 μm but retains only 60% ofWBCs. Because the cells lost in the 32-μm gap array are granulocytes andlymphocytes that are smaller than the reported CTC sizes, we selectedthis array for the CTC isolation system.

The second-stage herringbone chips were made of PDMS bonded to glasssubstrates using soft lithography techniques as previously describedabove. The chip surface was functionalized with anti-CD41 antibody(Abcam) using avidin-biotin chemistry.

The results are shown in FIG. 5 in the form of a graph showing (on theleft-hand side) CTC counts of 32 blood samples from metastatic lungpatients that were captured with EpCAM and CD41 antibodies respectively.All cells captured on the chip were processed with a four-color stainingassay for simultaneous CTC identification and platelet characterization.The captured cells that were positive for tumor markers (EpCAM,cadherin-11) and DAPI while negative for the hematopoietic markers(CD45) were scored as CTCs. Reliable CTC capture has been achieved in 21of 32 cases (66%), with counts ranging from 0.4 to 8.5 CTCs/mL.

In comparison, in-parallel CTC capture on microfluidic devicesfunctionalized with EpCAM antibodies revealed consistently lowerpositive hits (FIG. 5, right hand side). The higher CTC counts fromplatelet-targeted approach are due to the capability of the presentmethods to capture lung CTCs that might lose their epithelial naturethus difficult to target by EpCAM antibodies.

The platelets were stained by CD61 antibodies to characterize theirdistribution around CTC surface. The CTCs captured using the new systemexhibit a wide range of platelet coverage, including a subpopulation ofCTCs that were completely coated with platelets/fibrin. Theseplatelet-covered CTCs have been hypothesized as high metastaticpotential precursors because of platelet-enhanced tumor-cell survivaland proliferation, but are very difficult to capture by conventionalpositive selection methods targeting tumor surface antigens. Thus, thenew systems and methods provide a new and improved technique to capturethese CTCs.

Example 2—Isolation of Different Types of Cancers

The microfluidic platform was extended to isolate CTCs from differenttypes of cancer patients with both epithelial (breast) andnon-epithelial (melanoma) tumor origins. The breast CTCs were identifiedwith the same protocol as lung patient samples, while melanoma CTCs werestained with an antibody cocktail that targets melanoma specificantigens (CSPG4, MCAM, TYRP1, and α-SMA) as previously reported.Preliminary results have demonstrated reliable CTC capture for bothcancer types (3 of 5 cases for breast sample, counts ranging from 0.9 to2.7 CTC/mL; 5 of 6 cases for melanoma sample, counts ranging from 1.4 to17 CTC/mL). Similar to the lung cancer results, all the CTCs capturedwith current approach are associated with different extent of plateletcoverage.

As discussed above, the lung and breast CTCs were identified withEpCAM/Cadherin-11 (green) as epithelial and mesenchymal markers, whilethe melanoma CTCs were stained with a cocktail of CSPG4, MCAM, TYRP1,and α-SMA (green) antibodies. WBCs and platelets were stained with CD45(red) and CD61 (gold) respectively. Microscope imaging clearly showedeach type of CTC was captured (results not shown).

Thus, the system tested is capable of reliable capture of CTCs in theform of single cells or clusters from metastatic cancer patients withboth epithelial (lung, breast) and non-epithelial (melanoma) tumororigins. These results indicate that the platelet-targeted capture ofCTCs is effective for multiple types of cancer.

Example 3—Anticoagulant and CTC Purity

A key issue that potentially limits the performance of theplatelet-targeted approach is the relatively low purity of CTCs comparedwith other technologies. A large number of contaminating WBCs (>10⁵/mL)has been observed on chip, making the downstream analysis verychallenging. Staining with platelet-specific antibodies revealed thatmost of the captured WBCs were also coated with platelets. The formationof these so-called platelet-leukocyte aggregates (PLAs) originates fromspontaneous platelet activation and the consequent expression ofp-selectin, which will then bind to PSGL-1 receptors on leukocytesurface. PLAs are not removed by size-based sorting, and are captured byanti-platelet antibodies together with platelet-associated CTCs.

To reduce WBC contamination and improve CTC purity, we tested a varietyof platelet inhibitors, such as theophylline, adenosine, dipyridamole,Argatroban and prostaglandin I2, for blood stabilization. EpCAM-basedcapture was used as a control to evaluate the number of WBCs per mL ofblood sample. These results are shown on the left column of FIG. 7.EpCAM-based capture generally resulted in less WBC contamination, thoughthis method of CTC isolation suffers from a number of deficienciesdiscussed above. Platelet-based capture was tested using a variety ofplatelet inhibitors, with the results shown in the right column of FIG.7 (filled in circles represent untreated samples. The combination ofEDTA with prostaglandin I2 (shown as open triangles on the graph) wasfound to be effective in inhibiting the PLA formation, and reduced thenumber of contaminating WBCs by 90% (FIG. 7).

To completely inhibit the PLA formation, the blood sample was fixed inCyto-Chex® BCT tubes for 24 hours before processing, which yielded thebest CTC purity for platelet-based capture. These samples were referredto “fixed blood” (open circles).

FIGS. 8A and 8B show heat maps of microfluidic devices containingindividual captured WBCs (dark spots) mapped to their relative positionon the microfluidic device. There was a strong decay pattern with themajority of captured cells positioned at the inlet of the device withuntreated blood samples, suggesting specific interaction between WBCsand CD41 antibodies through surface platelets (FIG. 8A). In contrast,heat maps of prostaglandin I2-treated blood samples displayedsignificantly fewer captured cells, distributed randomly throughout thedevice (FIG. 8B).

Furthermore, by gently fixing the blood sample with commercial availableblood stabilization solutions (e.g., Cyto-Chex® BCT tube from Streck),PLA formation can be mostly suppressed during processing because of thecomplete platelet deactivation, giving rise to substantially improvedCTC purity that is comparable to the conventional EpCAM-targetedcapture. The CTC capture was not affected in these studies as the TCIPAoccurred under in-vivo conditions and those platelet-associatedaggregates were already formed before blood collection.

This solution to platelet-WBC aggregates improves the results of thetwo-stage microfluidic platform designed to achieve platelet-targetedCTC capture.

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1-22. (canceled)
 23. A two-stage microfluidic system for isolatingplatelet-associated nucleated target cells from a sample fluidcomprising: a first chamber comprising a microchannel having an inlet, awaste outlet, a product outlet, and an array of microposts arrangedbetween the inlet and the outlets, wherein the microposts are arrangedin rows and spaced apart by a distance that enables red blood cells andunbound platelets to flow through the device to a waste outlet and tocause platelet-associated nucleated target cells to be laterallydisplaced by the array of microposts to a product outlet, wherein themicroposts in each subsequent row are offset laterally from micropostsin a previous row by a distance less than the spacing between themicroposts within the row; a second chamber comprising a microchannelhaving an inlet and an outlet, wherein fluid flows from the inlet to theoutlet through the microchannel, and binding moieties fixed to at leastone internal surface of the microchannel, wherein the binding moietiesspecifically bind to platelets; and a fluid connection between theproduct outlet of the first chamber and the inlet of the second chamber.24. The system of claim 23, wherein the microposts are spaced apartwithin a row by a distance of about 30 microns to about 60 microns, andsubsequent rows are spaced apart from a previous row by a distance ofabout 5 microns to about 15 microns.
 25. The system of claim 23, whereinthe first chamber and the second chamber are both located on a singlesubstrate.
 26. The system of claim 23, wherein the first chamber and thesecond chamber are located on separate substrates and are in fluidconnection via the conduit.
 27. (canceled)
 28. The system of claim 23,wherein the binding moieties are bound to nanostructures that comprise afirst member of a binding pair, wherein one or more internal surfaces ofthe second chamber are bound to a layer of gelatin functionalized with aplurality of second members of the binding pair, and wherein thenanostructures are bound to a top layer of the gelatin by a bindinginteraction of the first and second members of the binding pair.
 29. Thesystem of claim 23, wherein the binding moieties comprise antibodiesthat bind specifically to platelets.
 30. A microfluidic system forisolating platelet-associated nucleated target cells from a bloodsample, the system comprising: a blood debulking chamber configured toremove a number of red blood cells and unbound platelets from the bloodsample; a cell capture chamber comprising a microchannel having an inletand an outlet, wherein sample fluid flows from the inlet to the outletthrough the microchannel, and binding moieties fixed to at least oneinternal surface of the microchannel, wherein the binding moietiesspecifically bind to platelets; and a fluid connection between theproduct outlet of the blood debulking chamber and the inlet of the cellcapture chamber.
 31. The system of claim 30, wherein the blood debulkingchamber comprises curved microfluidic channels to differentially focusand sort cells based on their sizes by centrifugal or inertial forces.32. The system of claim 30, wherein the blood debulking chambercomprises a hydrophoretic filtration system configured to sort cells ofdifferent sizes.
 33. The system of claim 30, wherein the blood debulkingchamber comprises an acoustic standing wave system configured to sortcells of different sizes.
 34. The system of claim 30, wherein the cellcapture chamber further comprises a plurality of grooves defined in andarranged on an internal surface of one or more walls, floor, and ceilingof the microchannel to create microvortices within the sample fluid. 35.The system of claim 30, wherein the blood debulking chamber comprises amicrochannel having an inlet, a waste outlet, a product outlet, and anarray of microposts arranged between the inlet and the outlets, whereinthe microposts are arranged in rows and spaced apart by a distance thatenables red blood cells and unbound platelets to flow through the deviceto a waste outlet and to cause platelet-associated nucleated targetcells to be laterally displaced by the array of microposts to a productoutlet, wherein the microposts in each subsequent row are offsetlaterally from microposts in a previous row by a distance less than thespacing between the microposts within the row.
 36. The system of claim34, wherein the microposts are spaced apart within a row by a distanceof about 30 microns to about 60 microns and subsequent rows are spacedapart from a previous row by a distance of about 5 microns to about 15microns.
 37. The system of claim 30, wherein the blood debulking chamberand the cell capture chamber are both located on a single substrate. 38.The system of claim 30, wherein the blood debulking chamber and the cellcapture chamber are located on separate substrates and are in fluidconnection via a conduit.
 39. The system of claim 30, wherein thebinding moieties are bound to nanostructures that comprise a firstmember of a binding pair, wherein one or more internal surfaces of thesecond chamber are bound to a layer of gelatin functionalized with aplurality of second members of the binding pair, and wherein thenanostructures are bound to a top layer of the gelatin by a bindinginteraction of the first and second members of the binding pair.
 40. Thesystem of claim 30, wherein the binding moieties comprise antibodiesthat bind specifically to platelets.